Influenza is a globally important respiratory pathogen that causes nearly annual epidemics and occasional pandemics. Recent spread of highly pathogenic avian influenza H5N1 viruses is a new threat that may lead to a deadly influenza pandemic. Influenza antiviral drugs are limited, although the need is urgent. Efficiency of progeny virus assembly and production is one of the critical factors of viral transmission and pathogenicity. However, the molecular mechanism of budding and virion formation is yet unclear. The goal of this research is to elucidate the mechanisms of virus assembly and progeny virion formation. Influenza viruses are assembled at the plasma membrane of infected cells by budding. Among the viral components, M1 proteins have been shown to be the key component in both bud formation, as well as the pinch-off that determines the size and shape of the virus particles. Our preliminary studies showed that the presence of specific NP prevented filament formation in infected cells, suggesting that an interaction between M1 and NP affects the budding and pinch- off process of influenza A viruses. In this research proposal, we hypothesize that appropriate M1-nucleocapsid (vRNP) interaction is required for efficient virus production and spherical virion formation. In Aim 1, we will identify M1-interacting domain(s) in NP and analyze the role of M1- NP interactions in viral assembly, production and morphology. Various recombinant influenza A viruses carrying chimeric or mutant NP genes will be generated by the reverse genetics system. In Aim 2, we will analyze the localization of vRNP in infected cells to determine if insufficient accumulation of vRNP at the plasma membrane due to the weak M1-vRNP interaction correlates with the morphological difference in progeny virions. We will utilize various approaches to chase vRNP distribution, including pulse-chase experiments and visualization of vRNPs in live cells using either FlAsH labeling or viral polymerase proteins fused to enhanced green fluorescence protein. Characterization of the role of M1-vRNP interaction in the virus assembly and budding processes is expected to unveil the mechanism of virion formation, which is a critical factor in virus transmission and pathogenicity. An understanding of the mechanism involved in efficient viral growth and spread should assist in the development of anti-viral strategies. PUBLIC HEALTH RELEVANCE: The efficiency of progeny virus assembly and production is one of the critical factors of transmission and pathogenicity of influenza A virus. However, the molecular mechanism of budding and virion formation is yet unclear. Studies proposed in this grant will unveil the process of virion formation and the role of M1-nucleocapsid interaction in virus assembly, thus yielding important implications for the design of antiviral reagents.